CSIRO PUBLISHING www.publish.csiro.au/journals/fpb Functional Biology, 2009, 36, 960–969

The shoot and root growth of and its potential as a model for and other crops

Michelle Watt A,C, Katharina Schneebeli A, Pan Dong A,B and Iain W. Wilson A

ACSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. BTriticeae Research Institute, Sichuan Agricultural University, Yaan, Sichuan 625014, . CCorresponding author. Email: [email protected]

Abstract. The grass genetic model Brachypodium ( (L.) Beauv., sequenced line Bd 21) was studied from germination to seed production to assess its potential as a phenotypic model for wheat (Triticum aestivum L.) and other cereal crops. Brachypodium and wheat shoot and root development and anatomy were highly similar. Main stem leaves and tillers (side shoots) emerged at the same time in both grasses in four temperature and light environments. Both developed primary and nodal axile roots at similar leaf stages with the same number and arrangement of vascular xylem tracheary elements (XTEs). Brachypodium, unlike wheat, had an elongated a mesocotyl above the seed and developed only one fine primary axile root from the base of the embryo, while wheat generally has three to five. Roots of both grasses could develop first, second and third order branches that emerged from phloem poles. Both developed up to two nodal axile roots from the coleoptile node at leaf 3, more than eight nodal axile roots from stem nodes after leaf 4, and most (97%) of the deepest roots at flowering were branches. In long days Brachypodium flowered 30 days after emergence, and root systems ceased descent 42 cm from the soil surface, such that mature roots can be studied readily in much smaller soil volumes than wheat. Brachypodium has the overwhelming advantage of a small size, fast life cycle and small , and is an excellent model to study cereal root system and function, as well as genes for resource partitioning in whole .

Additional keywords: Arabidopsis, architecture, genome, , photoperiod, root anatomy, tillering.

Introduction growth are similar to Brachypodium, their root–rhizosphere This paper presents a description of Brachypodium distachyon interactions may be similar, as well as root architectures for (L.) Beauv. (Brachypodium) sequenced line Bd 21 shoot and root different moisture and nutrient availabilities. Importantly, at systems from the seedling to grain filling stages. The aim was to maturity Brachypodium is much smaller than cereal crops, assess its potential to identify genes associated with the including other genetic models such as (Oryza sativa L.) development and function of wheat (Triticum aestivum L.) and (Zea mays L.) and it can be grown in controlled root systems. Brachypodium has been developed as a model conditions in small volumes of soil without greatly crowding for the genetics of cool climate grasses for fuel and crop roots and compromising their growth and function (Fig. 1). (Draper et al. 2001; Opanowicz et al. 2008; Vogel and Bragg Brachypodium presents a new opportunity to identify genes 2009). It has all the desirable features of a genetic model including associated with the growth and function of the roots of a small genome, short generation time between emergence and important cereal crops such as wheat, especially at more seed yield, and easy growth requirements. It is an annual, mature plant stages that are very important in field grown cereals. inbreeding and can be readily transformed (Vogel et al. 2006). (L.) is a small-sized genetic model, and The entire genome of diploid line Bd 21 is sequenced and has been the source of most genetic information for roots for publically available (J. Vogel, unpubl. data; http://www. nearly 20 years (Schiefelbein and Benfey 1991; Osmont et al. Brachypodium.org/, accessed 30 September 2009). 2007). However, it is a dicotyledon and member of the Brachypodium originates from the Fertile Crescent, the Brassicaceae family. Dicotyledons develop a tap root system geographic origin of wheat, and is considered a temperate with vascular cambia that produce new xylem and phloem with grass suited to cooler conditions (Vogel et al. 2009). On the development (Esau 1977). Monocotyledons like Brachypodium, phylogeny trees of the grass family , the and all members of the grass family, develop a ‘fibrous’ root Brachypodium is positioned with the temperate cereal genera system with no vascular cambia, and roots that emerge from the Triticum, Hordeum and Avena in the subfamily base of the embryo within the seed (primary or seminal axile (Kellogg 2001; Opanowicz et al. 2008). It can be expected roots), and from successive nodes along the stem (nodal axile that the environmental and soil conditions favourable to wheat roots) (Klepper et al. 1984; Wenzel et al. 1989; Hochholdinger

CSIRO 2009 Open Access 10.1071/FP09214 1445-4408/09/110960 Shoot and root growth of Brachypodium Functional Plant Biology 961

root systems traits in crops, especially deep in the soil, is challenging. In the field, this involves direct coring or time- consuming neutron moisture metre readings. By the time of flowering, many crop root systems extend more than 1 m below ground, and it is impossible to capture entire root systems in the field unless constrained in buried tubes. In controlled environments, root systems can be grown to maturity in root boxes greater than 1 m deep with clear faces, to map the spatial distribution of the entire root system to yield (Manschadi et al. 2006). However, these boxes are time- consuming and would be impractical to use on large numbers of lines in a genetics program. Most screening is performed on seedling roots in very small volumes or on agar or other artificial media in the laboratory, and it is unclear if these seedling traits contribute to mature root system traits and yield in the field. Here, we grew Brachypodium alongside wheat in soil in four environments and tracked the shoot and root systems growth and anatomical development, from germination to grain filling, to assess the potential of Brachypodium to identify genes regulating the root systems of wheat (cv. Janz), especially at adult plant stages.

Materials and methods Growing environments Brachypodium distachyon (L.) Beauv. (line Bd 21) seeds were kindly provided by Dr D. F. Garvin (USDA-ARS Plant Science Research Unit, University of Minnesota, MN, USA). Brachypodium was grown alongside wheat (Triticum aestivum L. cv. Janz) in the four environments described in Table 1 to compare their timing of root and shoot development, and to establish conditions that resulted in mature Brachypodium root systems that did not touch the bottoms of pots at grain filling. Three of the environments were short day, cooler conditions similar to those that occur in the field during wheat establishment and vegetative growth in Australia and other regions where wheat is grown in cool Fig. 1. Root systems of Triticum aestivum (cv. Janz) (left) and fi winter months; the fourth environment had long days and Brachypodium distachyon (line Bd 21) (right) grown to the ve-leaf stage fl in 50-cm tubes with a mixture of sand and soil in a growth cabinet in cool, warmer temperatures to promote more rapid owering (from short day conditions (environment 3 in Table 1). Bar = 10 cm. Vogel and Bragg 2009). For all experiments, seeds of Brachypodium and wheat were planted with the embryo of the seed facing the base of the pot, et al. 2004; Watt et al. 2008). The water transport through ~2 cm below the soil surface. For studies in the three short day dicotyledons and monocotyledons is different due, in part, to environments, the husks were removed from the Brachypodium their development and anatomy (Bramley et al. 2009). seeds and the seeds planted directly into soil and allowed to Other cereal models such as rice and maize are also very useful germinate in the conditions described above. For the long day for identifying root-associated genes (Hochholdinger et al. 2004; environment studies, the husks were not removed, the seeds Hochholdinger and Zimmermann 2008). However, they grow in planted, soil covered and pots vernalised at 7C in a cold room warm conditions. Rice, in particular, is adapted to waterlogged for 5 days, before transfer to the growth cabinet where the plastic conditions, unlike wheat. Their large adult size means both are cover was removed. Husk removal may have sped up germination difficult to screen in the laboratory to maturity. The root systems but the effect was marginal. Plants were grown in a soil that was a quickly reach the bottom of pots and become crowded and mix of 50% river sand and 50% recycled potting soil (organic impeded, and their deep root development and physiology matter, loam and sand supplemented with lime, 14.4% N, 6.6% P may not be relevant to that of field roots. Mature root system and 5% K). It was chosen because it drains well and washes off architecture and function are valuable to yield because they roots easily and was used to characterise wheat, (Hordeum determine the water that can be taken up around flowering and vulgare L.) and triticale (x Triticosecale) root systems in Watt grain filling, which contributes directly to final yield and crop et al. (2008). Plants were grown in PVC tubes of loosely packed water use efficiency (Passioura 1983; Manschadi et al. 2006; soil that were 7 cm wide, either 25 cm or 50 cm deep, and sealed at Kirkegaard et al. 2007). Directly selecting and validating mature the bottom with a Petri dish lid with holes for drainage. All plants 962 Functional Plant Biology M. Watt et al.

Table 1. Growing environments for Brachypodium distachyon (line Bd 21) and Triticum aestivum (cv. Janz)

Environment Daylength and conditions Details 1 Short day, outdoors 10–11 h sunlight in winter, 0À12C temperature range, half-strength Hoagland solution applied every 2 –3 days 2 Short day, glasshouse 10–11 h sunlight through glasshouse roof, 22/16C day/night temperatures, half- strength Hoagland solution applied every 2–3 days 3 Short day, growth cabinetA 10.5 h fluorescent bulb light, 300 mmol m–2 s–1 irradiance at leaf level, 15/15C day/ night temperatures, half-strength Hoagland solution applied every 2–3 days 4 Long day, growth cabinetA,B 20 h fluorescent bulb light, 300 mmol m–2 s–1 at leaf level, 24/18C day/night temperatures, tap water applied every 2–3 days AConviron CMP 2023 with cool-white fluorescent lights (Winnipeg, Manitoba, Canada). BBefore going into the growth cabinet, seeds were planted into pots, covered and left at 78C in a cold room for 5 days. were watered with tap water or half strength Hoagland’s nutrient transverse sections of pieces of root (~2 cm long) excised from the solution from the tops of the pots. Plants in environments 1 to 3 in basal regions of axile roots and from excised branch roots using the short day conditions received nutrients approximately twice the methods described in Watt et al. (2008). Briefly, the pieces a week; plants in environment 4 in long days did not receive were frozen in liquid nitrogen, mounted in low-temperature nutrients additional to those in the soil at sowing. mounting medium on stubs in the chamber of a cryo- microtome (Leica CM 1850; Leica Microsystems, Wetzlar, Shoot observations Germany), sections ~4 to 8 mm thick were made, mounted in Leaf and tiller (side shoot) emergence were scored every 2 to water on adhesive slides (Star-Frost Poly-L-lysine slides; ProSci 3 days according to the code developed by Zadoks et al. (1974). Tech, Thuringowa, Qld, Australia), dried on a warm plate, stained Flowering times were also noted, judged to be at the time that the with Rhodamine B (0.0005% w/w in water) and viewed with UV stems had elongated, heads had emerged partially and a few optics using a Leica DMR (Leica Microsystems) compound whitish stamens could be seen emerged from the florets. Leaf microscope. Rhodamine B is a metachromatic dye that fl lengths and plant heights were measured with a ruler. uoresces differently when excited by UV illumination depending on the wall compound it binds to. The method Brachypodium root system measurements results in sections with easily distinguishable central and peripheral xylem tracheary elements (XTEs). Their number Tubes were harvested at leaf 1, 2, 3, 4–5 and 7, and roots gently fi fi and arrangement were quanti ed to classify root types similar washed by rst saturating the soil in the pot, then removing the to the approach used for wheat, barley and triticale roots (Watt bottom and tilting the pots to slide out the soil and roots onto a et al. 2008). mesh tray to avoid breaking any fine roots. The remaining soil was washed away with a gentle spray of water and the entire shoot and intact root system floated in a large tray with water and a black Results background. The roots were gently disentangled and spread out to Shoots identify the positions of emergence of the axile roots around the The number and timing of emergence of leaves along the main seed and stem, and these were recorded in diagrams. The lengths stems of Brachypodium and wheat were nearly identical within of the axile roots and maximum root system depth were measured each of the four growing environments (Fig. 2). Tiller emergence with a ruler, noting which types of roots penetrated deepest in relative to main stem leaf emergence was also nearly identical fl fi plants at the onset of owering and at grain lling. The root for the two grasses in the shorter and longer day environments systems were then preserved in 50% ethanol until stained, (Fig. 3) with each developing four main stem tillers at similar fl scanned using an Epson 1680 atbed scanner (Regent stages of the development of six main stem leaves. The shoots of Instruments, Quebec, Canada) at 800 dpi resolution and the two grasses appeared similar with the exception that analysed for total root length (axile plus branch roots) using Brachypodium was shorter (~3.5 times shorter in the longer day, WINRhizo software (Regent Instruments) (Watt et al. 2005) or cool conditions; and ~2 times shorter in the long day, warmer used for the detailed morphological and anatomical studies conditions), more prostrate and the leaves were smaller. The time described below. to flowering strongly depended on the growing environment for both grasses. By 100 days post sowing, flowering had not Detailed morphological and anatomical studies of occurred in the grasses outdoors (environment 1, Table 1) and Brachypodium root systems they developed numerous secondary tillers. In the glasshouse The orders of branching along primary and nodal axile roots were (environment 2), Brachypodium flowered by 85 days and wheat scored under a dissecting microscope in a shallow dish of water. by 90 days post sowing. In the growth cabinet with cool, The diameters of different root types at their base and tip were short days (environment 3), Brachypodium and wheat measured whole-mounted on slides usinganeyepiece micrometer flowered by ~70 days post sowing. In contrast, the time to with brightfield optics with a compound microscope. Anatomies flowering in the long day conditions (environment 4) was of the different axile and branch root types were determined from much shorter in Brachypodium, which flowered ~30 days post Shoot and root growth of Brachypodium Functional Plant Biology 963

12.0 (a) 6.0 Short days

5.0 10.0

ber 4.0

8.0 3.0

Tiller num 2.0 6.0 1.0

4.0 0.0

Main stem leaf number

(b) 6.0 Long days 2.0

5.0 0.0 ber 4.0 0 10 20 30 40 50 60 70 80 90 Days after sowing 3.0

Fig. 2. Timing of leaf emergence of Triticum aestivum (cv. Janz) (dashed Tiller num 2.0 lines) and Brachypodium distachyon (line Bd 21) (solid lines) growing in four environments in soil (see Table 1 for details of environments). Diamonds, 1.0 cool, short days (10 h days; 0 to 12C) outside (environment 1); triangles, cool, short days (10 h days; 22/16C day/night) in a glasshouse (environment 2); 0.0 circles, cool, short days (10 h days; 15/15C day/night) in a growth cabinet (environment 3); squares, warm, long days (20 h days; 24/18C day/night) in a 0 1 2 3 4 5 6 7 8 9 10 growth cabinet (environment 4). Each point is the mean and standard error of Main stem leaf number five plants. Fig. 3. Timing of main stem tiller (lateral shoot) emergence relative to main stem leaf emergence of Triticum aestivum (cv. Janz) (dashed lines, open emergence; wheat flowered ~60 days post emergence. squares) and Brachypodium distachyon (line Bd 21) (solid lines, solid circles) Brachypodium line Bd 21 appeared to have strong growing in (a) short days (10 h days; 15/15C day/night) and (b) long days photoperiod sensitivity, although this shorter time to flowering (20 h days; 24/18C day/night) in a growth cabinet. Each point is the mean and in the long day conditions may have also depended on the cold standard error of five plants. treatment before transfer to the growth cabinet that was unique to this treatment. 4 days, wheat had germinated and grown three primary axile roots while Brachypodium had one; within 7 days, Brachypodium had one primary axile root while wheat had Root system development three to four. In the supplemental experiment in the long day, Brachypodium axile root development relative to leaf and warmer conditions (environment 4, Table 1) in soil, wheat tiller development is illustrated in Fig. 4. This sequence was developed five primary axile roots by 2 to 2.3 leaves, ranging observed in the short and long day conditions and is from 0.2 to 30.3 cm long, while Brachypodium had two to summarised from observations, drawing and measurements three leaves at the same time from sowing and had grown only from over 50 plants of each grown in eight separate one primary axile root 10.1–12.8 cm long. Brachypodium did experiments. The Brachypodium plants grown in the not develop a scutellar node axile root in these experiments, long days reached maturity, flowered and produced grain which can emerge in wheat from within the seed when the before the root systems reached the bottoms of the 50-cm plant is between leaf 1 and leaf 2 (Klepper et al. 1984; Watt tubes (Fig. 5), so most of the deep root observations are from et al. 2008). At leaf 3, up to two axile roots emerged from a mature plants in those conditions. node usually ~1 cm above the Brachypodium seed and below Brachypodium developed one primary axile root that the soil surface. Occasionally, no roots emerged from this node emerged from the base of the embryo at germination and and in some conditions, the node was just above or within the was the only axile root of the plant until leaf 3. This root seed. The anatomy at the base of the root was generally typical had no pith and generally one central XTE and four to eight of nodal roots, and comprised of pith surrounded by up to six peripheral XTEs (Fig. 6d). Wheat, as is well known, developed central XTEs and seven to nine peripheral XTEs (e.g. Fig. 6e). three to five primary axile roots from the base of the embryo. But occasionally no pith was present at the base and this root This difference was observed in all experiments, including two appeared as a primary axile root. We were unclear which stem supplemental experiments to confirm that the difference did not node this root emerged from, and although it may be the node depend on growing conditions. At 15C in the dark on moist associated with leaf 1, we think likely it is the node associated filter paper, Brachypodium germinated within 2 days growing with the coleoptile and term these roots coleoptile node axile a primary axile root from the base of the embryo; within roots, similar to those in wheat. Cross-sections above this node 964 Functional Plant Biology M. Watt et al.

L5 L6 L7 L4 L3 T4 L1 T3 T2 L2 T1

CNR LNR

seed

PR

Leaf 1 Leaf 3 Leaf 4 Leaf 5 Leaf 6, 7

Fig. 4. Axile roots of Brachypodium distachyon at different stages of main stem leaf (L) and tiller (T) development. PR, primary (seminal) axile root; CNR, coleoptile node axile root; LNR, leaf node axile roots. Line indicates soil surface. Branch roots are not drawn. First, second and third order branch roots could develop on all three types of axile roots. Summarised from observations from over 50 plants in eight separate experiments in short and long day conditions. Flowering commenced around leaf 6 or 7 in the warm, long days conditions and a head is drawn here on the elongated main stem only.

Days from coleoptile emergence with a central xylem tracheary element (XTE) (Fig. 6d). This 0 10 20 30 40 50 elongated internode is likely a mesocotyl which did not 0 3.0 2to3 4to5 6to7leaves 6to7leaves elongate in wheat in the experimental conditions in this leaves leaves Anthesis Grain filled )

–1 study. By leaf 5, axile roots had emerged from the stem 10 2.5 nodes associated with the main stem leaves, and the base of these roots had central piths with four to seven central XTEs 20 2.0 and 8 to 15 peripheral XTEs (Fig. 6e). By leaf 6 to 7, many roots had emerged from the stem nodes. In the long day 30 1.5 conditions, when flowering commenced at six to seven leaves, about nine leaf node axile roots were present per 40 1.0 plant (8.8 Æ 1.1, mean Æ s.d.; n = 5 plants). An example of the shifting contribution to total root system length from the Root system depth (cm) fi 50 0.5 primary to the nodal systems is quanti ed in Table 2. At leaf 6, the seminal and coleoptile node axile roots comprised a large Root system descent rate (cm d Root system descent rate 60 0.0 (80%) percentage of the total root length and the leaf node axile roots only 20% of the total root length; at leaf 8.5, the leaf Fig. 5. Root system depth (solid line) and rate of descent (dashed line) node axile roots comprise 56% of the total root length and the of Brachypodium distachyon grown in long day conditions in 50-cm deep number increased from 8 to 22. The primary axile root was and 7-cm wide tubes of soil. (mean and s.d. of four to six plants per harvest noticeably finer (mean diameter 220 mm) than the nodal axile time). roots (mean diameter 361 mm for coleoptile nodal roots; 354 mm for leaf node roots; Table 3), and by leaf 3, had indicated stem tissue with vascular bundles around a pith of many long first order branch roots, plus second parenchyma cells (Fig. 6a). Cross-sections through the node and occasionally third order branch roots. It contributed a (Fig. 6b) and below the node (Fig. 6c) revealed complex large root length with high specific root length for vascular arrangements of cells in transition from the Brachypodium growth and functioning until about leaf 4. arrangement in the stem above with discrete vascular Between leaf 5 and leaf 7, nodal system growth relative to bundles, and that in the seminal axile root below the seed, the primary root was rapid (Fig. 4). Shoot and root growth of Brachypodium Functional Plant Biology 965

C E

p p

(a) (e)

C C p

E E (b) (f ) (g )

C E

(c ) (h )

C C E

E

(d ) (i )

Fig. 6. Cross-sections through various parts of Brachypodium distachyon.(a–d) Sequence from stem to primary axile root. (a) Stem above coleoptile node shown in (b). (b) Coleoptile node between mesocotyl below in (c) and stem above in (a). A coleoptile node axile root is emerging from the node (star). (c) Mesocotyl above seed. (d) Basal region of primary axile root, below the seed. (e) Basal region of nodal axile root. ( f ) Branch root with seven XTEs (xylem tracheary elements) and the largest and highest number of XTEs in branch roots. (g) Branch root with one XTE and the simplest branch root observed. (h) Node axile roots with a branch root emerging (star) from the phloem (black arrowhead) pole. (i) Branch root with emerging branch root (star) from the phloem pole. Bar = 50 mminall images. Long white arrow indicates a central XTE. Arrowheads indicate phloem cells. C, cortex; E, endodermis; P, pith. 966 Functional Plant Biology M. Watt et al.

Table 2. Total root lengths of adult Brachypodium plants and the contributions of the primary and nodal systems Roots washed from soil, stained, scanned and analysed for total root length (axile and branch roots) as described in the text. Plants grown for 42 days in short day conditions in a growth cabinet; plant 1 in 25-cm long tubes; plant 2 in 50-cm long tubes

Plant Main Total root Primary axile root Coleoptile node axile root Leaf node axile roots stem system Total % Number Total % Number Total % leaves length length total root axile length total root axile length total root (cm) (cm) system roots (cm) system roots (cm) system 1 6 3137 1285 41 1 1198 38 8 654 21 2 8.5 6233 1273 20 2 1485 24 22 3475 56

All Brachypodium axile roots had the potential to develop Discussion fi rst, second and third order branch roots that ranged in diameter The similarities and differences between Brachypodium and m from 47 to 375 m (Table 3). These belonged to one of seven wheat root systems are summarised in Fig. 7. Key similarities types based on their stele anatomy and number and arrangement are that Brachypodium and wheat root systems had: (1) the of XTEs. They ranged from the most complex type with seven same types of axile roots; (2) nearly identical timing of main XTEs (Fig. 6f ) to the simplest type with one central XTE stem leaf and tiller emergence and timing of emergence of (Fig. 6g). Branch roots emerged from the phloem poles from axile roots; (3) very similar branch root types, sharing five axile (Fig. 6h) and branch roots (Fig. 6i). The different orders of types, with Brachypodium having two extra types; and branch root could be one of the seven types of branch root, (4)deeprootscomprisingofmainlybranchroots.The regardless of the axile root type. cereals had the same vascular anatomies especially the number and arrangement of the XTEs in axile and branch Deepest roots around flowering and grain filling roots. Branch roots in Brachypodium emerged from the In the longer day conditions that promoted quicker flowering, the phloem poles, in contrast to Arabidopsis where they root systems ceased descent around 42 cm from the soil surface emerge from the xylem poles. It is very useful that the (Fig. 5). The root systems descended initially at 0.86 cm per day vascular anatomies of the axile and branch roots until plants had four to five leaves, then descended at more than are common to wheat and to maize (compare with reports double that rate at 2.45 cm per day, to leaf 6 to 7 and flowering then in Hoppe et al. 1986; Varney et al. 1991; Watt et al. 2008). greatly decreased descent rate during grain filling to 0.15 cm This provides opportunities to understand water and per day (not significant). On average from the beginning to the end carbohydrate flow through the entire and intact root of vegetative growth, the root system descended at ~1.5 cm systems, relative to shoot development in grasses and to per day but the rate varied through plant growth. The bottom third of the root systems of plants post flowering weredominatedbybranchrootswhichmadeup97Æ 3% (mean Æ s.d., n = 7 plants) of the total roots present. First order branch roots made up 58 Æ 30% of total roots; second order branch roots made up 39 Æ 33% of the total roots; third order roots were not observed in deep layers. The deepest axile roots were a mixture of the primary root and coleoptile and leaf node axile roots (ratio of ~1 primary axile root to 4 nodal axile roots). M

Table 3. Diameters (mm) of root types of Brachypodium distachyon grown in soil Measurements from three plants grown in soil, from three seminal roots, four coleoptile node roots and eight leaf node roots. Diameters measured under a microscope using an eye piece micrometer of whole mounted roots. Mean and range (in brackets); n =1–15 measurements per root type Fig. 7. The axile roots of wheat (from Klepper et al. 1984; Watt et al. 2008; Primary root Coleoptile node Leaf node root and this study) and Brachypodium(line Bd 21; the present study) at the leaf (L) root stage at which they emerge. Branch roots are not drawn. Both can form first, second and third order branches on all axile roots. Solid lines, primary axile Axile 220 (138–280) 361 (162–650) 354 (192–562) roots (PR); large dashed lines, coleoptile node axile root (CNR); intermediate First order branch 102 (63–117) 166 (88–320) 190 (83–375) dashed line, scutellar node root (SNR) in wheat only; small dashed lines, leaf Second order branch 82 (67–100) 96 (62–130) 108 (47–242) node axile roots (LNR); M, a mesocotyl between seed and coleoptile node is Third order branch 75 (70–80) 103 (103) 95 (62–142) elongated in Brachypodium. Line indicates soil level. Shoot and root growth of Brachypodium Functional Plant Biology 967 contrast those with the dicotyledon genetic model members of the Bromus genus (Kellogg 2001; Bossolini Arabidopsis. et al. 2007). A notable difference between Brachypodium and the The mature root systems of wheat and Brachypodium were small grain cereals wheat, barley, triticale and (Avena nearly identical and grew to maturity within 50-cm tubes, sativa L.) is that the young root systems of B. distachyon line enabling intense study to grain filling. This provides an Bd 21 are much simpler with fewer numbers and types of opportunity to use Brachypodium to identify mature root axile roots. Brachypodium had one primary axile root from system developmental and functional genes in wheat. The the seed, did not produce a scutellar node axile root and the pattern of penetration of the Brachypodium roots was of development of axile roots from the node above the seed was interest, as it was slower initially, and then sped up greatly slightly delayed compared with that of the wheat coleoptile until flowering (Fig. 5). The pattern of descent was similar to node roots, or may have been associated with leaf one main that obtained from corings of root system depth of wheat in stem node. Brachypodium may have genes regulating early the field over a season (see fig.4inGregoryet al. 1978). root types that are more closely related to the warm climate They report a two-phase penetration rate; the first, between cereals, rice, maize, (Sorghumm bicolour L.), pearl the surface and 1 m depth, was three times slower than the (Pennisatum glaucum L.)andtheforagegrasses second, between 1 and 2 m depth. They suggest that this may such as smooth bromegrass (Bromus inermis Leyss), which be due to the winter and spring seasons. Kirkegaard and all generally have one primary axile root (Hoshikawa 1969; Lilley (2007) reviewed root system depth measurements Wenzel et al. 1989; Ries and Hofmann 1991; Hochholdinger across southern Australia and report a mainly linear pattern et al. 2004; M. Watt, unpubl. data). Brachypodium is of root system descent for wheat. However, penetration was currently positioned on phylogeny trees close to maize and slightly quicker earlier in the winter during early crop growth, sorghum and Bromus spp. (Kellogg 2001; Opanowicz et al. between 0.3 and 0.8 m, and slower later in the season between 2008, see fig. 7 therein). However, axile root number can vary 0.8 and 1.4 m (see fig. 3 in Kirkegaard and Lilley 2007). within species including wheat and maize (reviewed in Working in controlled environments, Derera et al. (1969) O’Toole and Bland 1987) and within Brachypodium. report a three-phase penetration pattern in wheat root systems; Hoshikawa (1969) conducted a survey of the seedling the first slow, to 0.2 or 0.3 m; the second rapid, to 1 m, and roots of over 219 species within 88 grass genera and the third slow, but with rapid nodal axile root growth and reported that the Brachypodium examined had branching. Brachypodium offers an opportunity to better more than one primary axile root. Numbers of axile roots understand root system growth through time and shoot at the three-leaf stage in Brachypodium and cereal crops may development, and variation due to and field be due to seed or embryo size. Mac Key (1978) reported one variability which has long been poorly understood for plant seminal root in the primitive wheat Aegilops mutica,uptosix roots. in Triticum durum L. and suggested more early axile roots The mature root systems of Brachypodium were may be a result of selection in cultivation and breeding dominated by branch roots which made up 97% of the programs for larger seeds. The low number of seedling total roots present. This is identical to the composition of axile roots in B. distachyon line Bd 21 is an opportunity deep field-grown wheat roots, quantified using the anatomy to identify the genes associated with greater numbers of early of cored, washed and sectioned roots (Watt et al. 2008). seedling roots among the diverse Brachypodium genotypes The axile Brachypodium roots were a mixture of the primary now available (Vogel et al. 2009). This will enable us to root and coleoptile and leaf nodal roots and these were better understand their contribution relative to nodal roots to readily identified because it was possible to follow the mature plant function. deep axile roots to their position of emergence after Interestingly, Brachypodium had an elongated internode washing from soil. The finding of nodal roots among the between the seed and the coleoptile node in the soil-grown bottom 30% of the root system contradicts our previous conditions here when planted 2 cm below the soil surface findings with field-grown wheat (Watt et al. 2008) and (Fig. 7). The elongated internode in Brachypodium is likely a those of Weaver (1926). Araki and Iijima (2001) reported mesocotyl and the anatomy is transitional between that of the that the nodal roots from the coleoptile and stem nodes of leaf stem above and that of the primary axile root below 1 and 2 can grow as long as the primary axile roots in certain (Fig. 6a–d) (see also Raju and Steeves 1998). Mesocotyls wheat cultivars. Weaver (1926) followed axile roots from the are reported in other grasses including maize and oats seed to depth in the field and report that the nodal roots did (Radford and Key 1993) and in smooth bromegrass (Ries not reach the deepest layers. Weaver (1926) may have and Hofmann 1991). Wheat did not have an elongated mistaken the scutellar, coleoptile and leaf 1 nodal roots, internode in this position in our experiments. A mesocotyl which emerge close to the seed if planted at a shallow may be a difference between Brachypodium and wheat, depth, with the primary axile roots. Brachypodium can be although wheat genotypes may elongate a mesocotyl under used to quantify the relative contribution of the primary and certain conditions such a very deep planting. Hoshikawa nodal systems at different depths in freely-growing root (1969) classified Brachypodium as having a ‘more or less’ systems to yield. This is an area of current investigation elongated mesocotyl. The observations that Brachypodium using Brachypodium, tracking individual root axes and water readily develops a mesocotyl and only one axile primary root uptake through time to grain filling, using new non-invasive suggests that Brachypodium shares developmental pathways imaging phenotyping methods. The relative rates of growth of with the warm climate cereals maize and rice and with the different Brachypodium root axes with time and shoot 968 Functional Plant Biology M. Watt et al. development to grain filling is an important area for research HoshikawaK (1969) Underground organs of the seedlings and the systematics to improve root system genetics, coupled with studies to see if of Gramineae. Botanical Gazette (Chicago, Ill.) 130, 192–203. similar processes can be enhanced in wheat. doi: 10.1086/336490 Kellogg EA (2001) Evolutionary history of the grasses. Plant Physiology 125, 1198–1205. doi: 10.1104/pp.125.3.1198 Conclusions Kirkegaard JA, Lilley JM (2007) Root penetration rate – a benchmark to Brachypodium root systems had a high degree of developmental identify soil and plant limitations to rooting depth in wheat. Australian Journal of Experimental 47, 590–602. doi: 10.1071/ and anatomical similarity to wheat root systems, especially at the EA06071 adult plant stages. Its small stature and identical coordination Kirkegaard JA, Lilley JM, Howe GN, Graham JM (2007) Impact of subsoil between root and shoot development to wheat means the function water use on wheat yield. Australian Journal of Agricultural Research of genes regulating partitioning and water uptake of adult root 58, 303–315. doi: 10.1071/AR06285 systems can be studied intensely. Because Brachypodium had Klepper B, Belford RK, Rickman RW (1984) Root and shoot development fewer numbers of seedling axile roots, there are opportunities to in winter wheat. Agronomy Journal 76, 117–122. select for more of these root types among the diverse genetic Mac Key J (1978) Wheat domestication as a shoot : root interrelation resources available in Brachypodium (Vogel et al. 2009). process. In ‘Proceedings of 5th International Genetics Symposium, ’ – Brachypodium is likely to speed up cereal root system genetic New Delhi. Vol 2 . (Ed. S Ramanujam) pp. 875 890. and functional discoveries. Manschadi AM, Christopher J, deVoil P, Hammer GL (2006) The role of root architectural traits in adaptation of wheat to water-limited environments. Functional Plant Biology 33, 823–837. doi: 10.1071/ Acknowledgements FP06055 O’Toole JC, Bland WL (1987) Genotypic variation in crop plant root We are grateful to Dr Dave Garvin for supplying the Brachypodium systems. Advances in Agronomy 41,91–145. doi: 10.1016/S0065- distachyon line Bd 21 seed, Dr John Vogel for advice on growing 2113(08)60803-2 conditions and Drs Frank Hochholdinger, Margaret McCully and Richard OpanowiczM,VainP,DraperJ,ParkerD,DoonanJH(2008) Richards for valuable suggestions to the manuscript. This study was funded in Brachypodium distachyon: making hay with wild grass. Trends part by a CSIRO Julius Award to MW and KS, a CSIRO-Ching Scholarship to in Plant Science 13,172–177. doi: 10.1016/j.tplants.2008.01.007 PD and the Grains Research and Development Corporation (GRDC). 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